Neurotrophic Tyrosine Kinase Receptor Research Articles

Phase separation underlies signaling activation of oncogenic NTRK fusions

NTRK (neurotrophic tyrosine receptor kinase) gene fusions that encode chimeric proteins exhibiting constitutive activity of tropomyosin receptor kinases (TRK), are oncogenic drivers in multiple cancer types. However, the underlying mechanisms in oncogenesis that involve various N-terminal fusion partners of NTRK fusions remain elusive. Here, we show that NTRK fusion proteins form liquid-like condensates driven by their N-terminal fusion partners. The kinase reactions are accelerated in these condensates where the complexes for downstream signaling activation are also concentrated. Our work demonstrates that the phase separation driven by NTRK fusions is not only critical for TRK activation, but the condensates formed through phase separation serve as organizational hubs for oncogenic signaling.

Uterine Neurotrophic Tyrosine Receptor Kinase Rearranged Spindle Cell Neoplasms: Three Cases of an Emerging Entity.

Uterine sarcomas are rare; most are either smooth muscle or endometrial stromal in origin. Recent molecular advances have identified several, genetically defined entities with specific morphologic, clinicopathological associations, and therapeutic options. We report 3 cases of uterine neurotrophic tyrosine receptor kinase ( NTRK )-rearranged spindle cell neoplasms," currently classified as "emerging entities" in the WHO Classification of Female Genital Tract Tumors, 2020, Fifth Edition. The affected patients were 32, 34, and 42 years of age. Two patients presented with vaginal bleeding; the third presented with a cervical mass found incidentally during laparoscopy for an ectopic gestation. All 3 tumors were polypoid masses that distorted the cervix. Microscopically, they comprised cellular, fascicular, and storiform, plump spindle cells, with occasional rounded cells, and frequent mitoses (4-48/10 high power fields) in a myxoid stroma. All 3 cases showed entrapment of benign cervical glands. Inflammatory cell infiltrates, including plasma cells, were noted in all 3 tumors. One case had tumor cell necrosis, osteoid-like material, and osteoclast-like giant cells and showed lymphovascular invasion. Immunohistochemically, our cases showed patchy S100 (2/3) and CD34 (3/3) positivity. CD10 was positive in 2/3 cases. 3/3 cases showed pan-tropomyosin receptor kinase positivity (cytoplasmic). The NTRK -translocations demonstrated were: NTRK1::TMP3, NTRK1::TPR, and NTRK3::SPECC1L . Two of the patients had extensive disease and underwent chemotherapy. Larotrectinib was approved for one patient who demonstrated a striking reduction in tumor volume upon initiation of this treatment.

The Safety Profiles of Two First-Generation NTRK Inhibitors: Analysis of Individual Case Safety Reports from the FDA Adverse Event Reporting System (FAERS) Database.

The first-generation tropomyosin receptor kinase (TRK) inhibitors, larotrectinib and entrectinib, represent exciting new developments in cancer treatment that offer relevant, rapid, and long-lasting clinical benefits. Larotrectinib and entrectinib are recommended as first-line treatments for locally advanced or metastatic non-small cell lung cancer (NSCLC) patients with positive TRK gene fusions. In this study, using the U.S. Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) database between 2019 and 2022, a retrospective analysis was conducted to evaluate the safety profiles of these drugs. During our study period, 807 individual case safety reports (ICSRs) related to larotrectinib or entrectinib were retrieved from the FAERS database, of which 48.7% referred to females and 24.7% referred to adult patients (18-64 years) with a median age of 61.0 years. A total of 1728 adverse drug reactions (ADRs) were identified. The most frequently reported ADRs were dizziness and pain, which belong to the System Organ Classes (SOCs) "nervous system disorders" and "general disorders and administration site conditions". Regarding all ADRs, the median time to onset was 37.0 days for larotrectinib and 12.0 days for entrectinib. No evident safety concerns emerged in the long-term safety profiles (>365 days). Only 18 ICSRs were related to pediatric populations (≤16 years), of which 94.0% of the ICSRs were related to larotrectinib. The median age was 10.5 years, while most patients were female (44.4%). Our results show favorable risk-benefit profiles for larotrectinib and entrectinib. Considering the increased use of neurotrophic tyrosine receptor kinase (NTRK) inhibitors, continuous safety monitoring of larotrectinib and entrectinib is required for the detection of possible new adverse drug reactions.

Corticosterone effects on postnatal cerebellar development in mice

Glucocorticoids administered early in infancy can affect the architectonic organization of brain structures, particularly those with a postnatal development and resulting in long-term deficits of neuromotor function and cognition. The present study was undertaken to study the effects of daily corticosterone (CORT) injections at a pharmacological dose from postnatal days 8–15 on cerebellar and hippocampal development in mouse pups. Gene expression status for trophic factors involved in synaptic development and function as well as measures of layer thickness associated with cytochrome oxidase labelling were analyzed in the hippocampus, hypothalamus, and specific cerebellar lobules involved in motor control. Repeated CORT injections dysregulated the HPA axis with increased Crh and Nr3c1 mRNA levels in the hypothalamus and a resulting higher serum corticosterone level. The CORT treatment altered the morphology of the hippocampus and down-regulated gene transcription for corticotropin-releasing hormone (Crh) and its type-1 receptor (Crhr1), glucocorticoid receptor (Nr3c1), and brain-derived neurotrophic factor Bdnf and its receptor Ntrk2 (neurotrophic receptor tyrosine kinase 2). Similar mRNA expression decreases were found in the cerebellum for Crhr1, Crhr2, Nr3c1, and Grid2 (glutamatergic δ2 receptor). Morphological alterations and metabolic activity variations were observed in specific cerebellar lobules involved in motor control. The paramedian lobule, normally characterized by mitotic activity in the external germinative layer during the second postnatal week, was atrophic but metabolically hyperactive in its granule cell and molecular layers. On the contrary, lobules with an earlier cell proliferation displayed neurogenesis but a hypoactivated granule cell layer, suggesting a developmental delay in synaptogenesis. The results indicate that glucocorticoid, administered daily during the second postnatal week modulated the developmental programming of the hippocampus and cerebellum. These growth and metabolic alterations may lead possibly to morphological and functional changes later in life.

NTRK fusion events and targeted treatment of advanced radioiodine refractory thyroid cancer

PurposePathogenic fusion events involving neurotrophic receptor tyrosine kinase (NTRK) have been described in ~ 2% of differentiated thyroid cancer (DTC). The selective tropomyosin receptor kinase (TRK) inhibitors entrectinib and larotrectinib have been approved in a tumor agnostic manner based on phase 1/2 clinical trials. In a real-world setting at five referral centers, we aimed to describe the prevalence of NTRK gene fusions and the efficacy and safety of TRK inhibitor treatment for non-medullary, advanced thyroid cancer (TC).MethodsA total of 184 TC patients with testing for NTRK gene fusions were included. Progression-free survival (PFS) and overall survival (OS) probabilities were estimated using the Kaplan–Meier method in six patients with NTRK fusion-positive TC who underwent TRK inhibitor therapy.Results8/184 (4%) patients harbored NTRK gene fusions. Six patients with radioiodine (RAI)-refractory TC harboring NTRK1 (n = 4) and NTRK3 (n = 2) gene fusions were treated with larotrectinib. Five patients (83%) had received ≥ 1 prior systemic therapy and one patient did not receive prior systemic therapy. All patients had morphologically progressive disease before treatment initiation. Objective response rate was 83%, including two complete remissions. Median PFS from start of TRK inhibitor treatment was 23 months (95% confidence interval [CI], 0–57.4) and median OS was not reached (NR) (95% CI, NR). Adverse events were of grade 1–3.ConclusionThe prevalence of NTRK gene fusions in our cohort of RAI-refractory TC is slightly higher than reported for all TC patients. Larotrectinib is an effective treatment option in the majority of NTRK gene fusion-positive advanced TC patients after prior systemic treatment and has a favorable safety profile.

New Era of Immune-Based Therapy in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (CC) accounts for approximately 20% of all biliary tract cancer (BTC) cases and 10-15% of all primary liver cancer cases. Many patients are diagnosed with unresectable BTC, and, even among patients with resectable BTC, the 5-year survival rate is approximately 20%. The BTC incidence rate is high in Southeast and East Asia and has increased worldwide in recent years. Since 2010, cytotoxic chemotherapy, particularly combination gemcitabine + cisplatin (ABC-02 trial), has been the first-line therapy for patients with BTC. In 2022, a multicenter, double-blind, randomized phase 3 trial (TOPAZ-1 trial) examined the addition of programmed death-ligand 1 immunotherapy (durvalumab) to combination gemcitabine + cisplatin for BTC treatment, resulting in significantly improved survival without notable additional toxicity. As a result of this trial, this three-drug combination has become the new standard first-line therapy, leading to notable advances in BTC management for the first time since 2010. The molecular profiling of BTC has continued to drive the development of new targeted therapies for use when first-line therapies fail. Typically, second-line therapy decisions are based on identified genomic alterations in tumor tissue. Mutations in fibroblast growth factor receptor 1/2/3, isocitrate dehydrogenase 1/2, and neurotrophic tyrosine receptor kinase A/B/C are relatively frequent in intrahepatic CC, and precision medicines are available that can target associated pathways. In this review, we suggest strategies for systemic pharmacotherapy with a focus on intrahepatic CC, in addition to presenting the results and safety outcomes of clinical trials evaluating immune checkpoint inhibitor therapies in BTC.

Unraveling the molecular landscape: a comparative analysis of PI3K and MAPK signaling pathways in plasmablastic lymphoma and diffuse large B-cell lymphoma with therapeutic implications

Plasmablastic lymphoma (PBL) is a rare and aggressive subtype of non-Hodgkin lymphoma that shares features with diffuse large B-cell lymphoma (DLBCL). While significant progress has been made in treating DLBCL, the prognosis for PBL remains poor, highlighting the need to identify new therapeutic targets. Using RNA expression analysis, we compared the expression of genes involved in the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways between PBL and DLBCL. We used critical PI3K (n=201) and MAPK (n=57) signaling probe sets to achieve this objective. Our results demonstrate unique molecular mechanisms underlying PBL pathogenesis compared to DLBCL, particularly within the PI3K and MAPK signaling pathways. We found that elevated STAT3 expression in PBL correlates with hyperactive MAPK and PI3K pathways, unlike DLBCL. Additionally, the hyperactivation of the PI3K signaling axis in PBL is unrelated to B-cell receptoror phosphatase and tensin homologactivity, indicating a distinct mechanism compared to DLBCL. Furthermore, we observed unique activation patterns in MAPK pathways between PBL and DLBCL, with PBL exhibiting high expression of the neurotrophic tyrosine kinase receptor (NTKR) family, specifically NTRK1 and NTRK2 genes, which have therapeutic potential. We also found that neither human immunodeficiency virus nor Epstein-Barr virus infection influences gene expression profiles linked to PI3K and MAPK signaling in PBL. These findings could lead to adapting targeted therapies developed for DLBCL to address the specific needs of PBL patients better and contribute to developing novel, targeted therapeutic strategies to improve patient outcomes.

Tyrosine kinase alterations in colorectal cancer with emphasis on the distinct clinicopathological characteristics.

Tyrosine kinase (TK) alterations, such as anaplastic lymphoma kinase (ALK) fusion, neurotrophic tyrosine receptor kinase (NTRK) fusion, c-ros oncogene 1 (ROS1) fusion and mesenchymal-epithelial transition factor (MET) exon 14 skipping, have been reported in colorectal cancers (CRC). We have previously reported CRCs with NTRK fusion among our cohort. However, their clinicopathological features have not been fully elucidated. Tissue microarray (TMA)-based immunohistochemistry (IHC) was performed on 951 CRC lesions from 944 patients. IHC was evaluated as positive or negative for ALK and ROS1 and 0 to 3+ for c-MET. For ALK and ROS1 IHC-positive cases, RNA-based imbalanced gene expression assays, Archer FusionPlex assays and reverse transcription-polymerase chain reaction (RT-PCR) followed by Sanger sequencing were performed. For c-MET IHC 3+ cases, RT-PCR followed by Sanger sequencing were performed. ALK IHC was positive in three cases (0.2%) and all showed imbalanced ALK gene expression. The following ALK fusions were confirmed: EML4 (exon 21)::ALK (exon 20), EML4 (exon 6)::ALK (exon 19) and HMBOX1 (exon 6)::ALK (exon 20). Two showed microsatellite instability-high/mismatch repair (MMR)-deficient, and all were located in the right colon. ROS1 IHC was positive in one case; however, imbalanced expression and ROS1 fusion was negative. Forty-two cases (4.4%) showed c-MET IHC3+. MET exon 14 skipping was confirmed in nine cases. All cases were microsatellite stable/MMR-proficient, and eight were located in the left colon and rectum. CRCs with these TK alterations had distinct clinicopathological features. Together with our previous study, 15 cases (1.6%) harboured targetable TK alterations (three NTRK fusion, three ALK fusion, nine MET exon 14 skipping).

Congenital Insensitivity TO Pain: A Case Report

Congenital insensitivity to pain or more scientifically Hereditary sensory and autonomic neuropathies (HSAN) is a rare genetic disorder which associates a sensory dysfunction with a varying degree of autonomic dysfunction. Due to the peripheral neuropathy, a decreased sensitivity or even complete anesthesia may be present resulting in, on the ophthalmological level, neurotrophic ulcers. We report the case of 2 sisters (JM and KM) presenting with HSAN with recurrent corneal ulcers. Unfortunately, genetic testing couldn’t be performed due to lack of means, but the clinical presentation and features were very favourable or even pathognomonic of this syndrome. The first cases or reported individuals presenting with congenital insensitivity to pain goes back to 1930’s. Five types of hereditary sensory and autonomic neuropathy have been identified according to age of onset of symptoms, clinical features and affected gene. HSAN type IV also known as congenital insensitivity to pain with anhidrosis (CIPA) is the second most common HSAN. It is caused by mutation in the NTRK1(Neurotrophic tyrosine kinase receptor type 1) (TRKA) gene located in chromosome 1 (1q21-q22). It is characterized by repetitive hyperthermic episodes in infancy, and mental retardation is usually present, as reported in our case. Clinical symptoms of pain insensitivity manifest as tongue, lip and fingers biting, and self-inflicted injuries. Congenital insensitivity to pain is a rare genetic syndrome characterized by an absence or an altered response to pain. Individuals with this syndrome can presented self-inflicted injuries and auto-mutilation leading in some cases to severe disabilities. Long-term visual prognosis in CIPA patients is not assessed and there’s an important lack of data regarding ocular manifestation of CIP syndrome.

Outcome of Children and Adolescents With Relapsed/Refractory/Progressive Malignancies Treated With Molecularly Informed Targeted Drugs in the Pediatric Precision Oncology Registry INFORM.

INFORM is an international pediatric precision oncology registry, prospectively collecting molecular and clinical data of children with recurrent, progressive, or very high-risk malignancies. We have previously identified a subgroup of patients with improved outcomes on the basis of molecular profiling. The present analysis systematically investigates progression-free survival (PFS) and overall survival (OS) of patients receiving matching targeted treatment (MTT) with the most frequently applied drug classes and its correlation with underlying molecular alterations. A cohort of 519 patients with relapsed or refractory high-risk malignancies who had completed a follow-up of at least 2 years or shorter in the case of death or loss to follow-up was analyzed. Survival times were compared using the log-rank test. MTT with anaplastic lymphoma kinase (ALK), neurotrophic tyrosine receptor kinase (NTRK), and B-RAF kinase (BRAF) inhibitors showed significantly improved PFS (P = .012) and OS (P = .036) in comparison with conventional treatment or no treatment. However, analysis of the four most commonly applied MTT groups, mitogen-activated protein kinase (MEK- n = 19), cyclin-dependent kinase (CDK- n = 23), other kinase (n = 62), and mammalian-target of rapamycin (mTOR- n = 20) inhibitors, did not reveal differences in PFS or OS compared with conventional treatment or no treatment in patients with similar molecular pathway alterations. We did not observe differences in the type of pathway alterations (eg, copy number alterations, single-nucleotide variants, InDels, gene fusions) addressed by MTT. Patients with respective molecular alterations benefit from treatment with ALK, NTRK, and BRAF inhibitors as previously described. No survival benefit was observed with MTT for mutations in the MEK, CDK, other kinase, or mTOR signaling pathways. The noninterventional character of a registry has to be taken into account when interpreting these data and underlines the need for innovative interventional biomarker-driven clinical trials in pediatric oncology.

Japanese Society of Medical Oncology/Japan Society of Clinical Oncology/Japanese Society of Pediatric Hematology/Oncology-led clinical recommendations on the diagnosis and use of immunotherapy in patients with high tumor mutational burden tumors

The development of novel antitumor agents and accompanying biomarkers has improved survival across several tumor types. Previously, we developed recommendations for tumor-agnostic treatments in patients with solid tumors with DNA mismatch repair deficient or neurotrophic receptor tyrosine kinase fusions. Recently, immune checkpoint inhibitors have shown efficacy in patient with tumor mutation burden-high (TMB-H) solid tumors and have been established as a third tumor-agnostic agent, making it necessary to develop the guideline prioritized for these patients. Clinical questions regarding medical care were formulated for patients with TMB-H advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by Japan Society of Clinical Oncology (JSCO), Japanese Society of Medical Oncology (JSMO), and Japanese society of pediatric hematology/oncology (JSPHO) voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and JSPHO, and the public comments among all societies' members was done. The current guideline describes three clinical questions and seven recommendations for whom, when, and how TMB should be tested, and what is recommended for patients with TMB-H advanced solid tumors. In this guideline, the committee proposed seven recommendations for performing TMB testing properly to select patients who are likely to benefit from immunotherapy.

Andrographispaniculata methanol extract suppresses the phosphorylation of ETV6‑NTRK3.

ETS variant transcription factor 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) fusions are typically found in rare diseases, such as primary renal fibrosarcoma (only six cases have been reported), secretory carcinoma of the breast and salivary gland (1 case), and AML (4 cases). Few cases have been reported, and expression of the EN gene fusion requires additional clinical data and fundamental research to be supported. The aim of the present study was to determine the inhibitory effect of Andrographis paniculata methanol extract (MeAP) on EN-related cell lines, IMS-M2 and BaF3/EN, as well as evaluate the mechanism of action. Vero cells were used as control cells. Trypan blue staining and MTT were used to evaluate the inhibitory effect of MeAP on tested cells. Western blotting and immunoprecipitation were used to detect the activation of EN after MeAP treatment. The IC50 values of MeAP were found to be 12.38±0.57 µg/ml (IMS-M2) and 13.06±0.49 µg/ml (BaF3/EN). MeAP was observed to inhibit cell proliferation in a time, dose, and cell density-dependent manner. The IC50 value for MeAP in Vero cells was markedly higher, at 109.97±4.24 (µg/ml), indicating a much less sensitive effect. Furthermore, MeAP treatment inhibited EN phosphorylation and induced apoptosis in these cells. Collectively, the present study demonstrated that MeAP has an oncogenic effect on EN fusion-positive cell lines, in particular.

Larotrectinib long-term efficacy and safety in adult patients (pts) with tropomyosin receptor kinase (TRK) fusion cancer.

3141 Background: Neurotrophic tyrosine receptor kinase ( NTRK) gene fusions are oncogenic drivers in a broad array of tumor types. Larotrectinib is a first-in-class, highly selective, central nervous system (CNS)-active TRK inhibitor approved for tumor-agnostic use in pts with TRK fusion cancer based on a rapid, robust, and durable objective response rate (ORR) in both adult and pediatric pts with various non-primary CNS cancers. Here, we report data on an expanded cohort of adult pts with TRK fusion cancer treated with larotrectinib. Methods: Adults with non-primary CNS TRK fusion cancer treated in three larotrectinib clinical trials (NCT02576431, NCT02122913, and NCT02637687) were included. Larotrectinib was administered at 100 mg twice daily in most pts. Responses were assessed per Independent Review Committee (IRC) using RECIST v1.1. Data cut-off: July 2022. Results: As of July 2022, there were 194 adult pts enrolled, with 180 eligible for efficacy analyses by IRC; 22 pts had known brain metastases at baseline. There were 24 different tumor types; the most common were lung (n=30; 15%), soft tissue sarcoma (n=30; 15%), thyroid (n=28; 14%), salivary gland (n=25; 13%), and colon (n=23; 12%). Median age was 57 years (range 19–90). The gene fusions involved NTRK1 (n=90; 46%), NTRK2 (n=7; 4%), or NTRK3 (n=97; 50%). Pts had previously received a median of one systemic therapy (range 0–10). ORR was 57% (95% CI 50–65): 29 (16%) complete response (including one pathological complete response), 74 (41%) partial response, 39 (22%) stable disease, 23 (13%) progressive disease, and 15 (8%) not evaluable. ORR in 22 evaluable pts with baseline CNS metastases was 68% (95% CI 45–86). Median time to response was 1.8 months, and median duration of response was 43.3 months (95% CI 29.2–not estimable [NE]) at a median follow-up of 32.3 months. Median progression-free survival was 24.6 months (95% CI 11.3–34.5) at a median follow-up of 28.5 months. Median overall survival was 48.7 months (95% CI 38.5–NE) at a median follow-up of 33.8 months. At data cut-off, 39 of the 73 pts who had disease progression continued treatment post-progression for ≥4 weeks. Treatment-related adverse events (TRAEs) were predominantly Grade 1/2. Grade 3/4 TRAEs occurred in 27 (14%) pts. Treatment discontinuation due to TRAEs was reported in one pt (increased alanine aminotransferase and aspartate aminotransferase). Conclusions: In this expanded dataset with extended follow-up, larotrectinib continues to demonstrate rapid and durable responses, extended survival, and a favorable safety profile in adults with TRK fusion cancer, including those with CNS metastases. These results support the wider adoption of next-generation sequencing panels that include NTRK gene fusions when testing pts with solid tumors; this ensures that pts are offered the right medicine for their disease as soon as possible. Clinical trial information: NCT02576431 , NCT02122913 , NCT02637687 .

Long-term efficacy and safety of larotrectinib in patients with tropomyosin receptor kinase (TRK) fusion lung cancer.

9056 Background: Neurotrophic tyrosine receptor kinase ( NTRK) gene fusions are oncogenic drivers in a variety of tumor types, including lung cancer. Larotrectinib is a highly selective, central nervous system (CNS)-active TRK inhibitor that demonstrated rapid, deep, and durable responses in patients (pts) with TRK fusion lung cancer (Drilon et al. ASCO 2022) and is considered a preferred first-line therapy option for these pts (Owen et al. J Clin Oncol 2022). We report updated data from a larger cohort with longer follow-up. Methods: Pts with TRK fusion lung cancer enrolled in 2 larotrectinib clinical trials (NCT02576431 and NCT02122913) were included in this analysis. Larotrectinib was administered at 100 mg twice daily, and response was assessed by independent review committee (IRC) per RECIST v1.1. Results: As of July 20, 2022, 30 pts with a median age of 55.5 years (range 25–81) with TRK fusion lung cancer enrolled, including 12 pts with CNS metastases at baseline. The gene fusions involved NTRK1 (n = 24; 80%) or NTRK3 (n = 6; 20%). Pts received a median of 2 prior lines of systemic therapies, with 20 pts (67%) receiving ≥2. Among 27 pts eligible for IRC assessment, the objective response rate (ORR) was 74% (95% CI 54–89): 3 complete responses, 17 partial responses (PR), 4 stable disease (SD), 2 progressive disease (PD), and 1 not evaluable. Among the 12 pts with baseline CNS metastases, the ORR was 67% (95% CI 35–90): 8 PR, 2 SD, and 2 PD. The median time to response for all responders was 1.8 months. Median duration of response (DoR) was 33.9 months (95% CI 9.5–not estimable [NE]); median follow-up was 22.9 months. Median progression-free survival (PFS) was 33.0 months (95% CI 11.3–NE); median follow-up was 24.7 months. Median overall survival (OS) was 39.3 months (95% CI 17.2–NE); median follow-up was 23.1 months. For the 12 pts with CNS metastases, the medians for DoR, PFS, and OS were 9.5, 9.9, and 19.4 months, with median follow-ups of 17.4, 19.3, and 22.2 months, respectively. Duration of treatment for all pts evaluable per IRC ranged from 0.3+ to 75.2+ months. At data cut-off, 10 pts had progressed, with 6 continuing treatment post-progression for ≥4 weeks due to continued clinical benefit. Treatment-related adverse events (TRAEs) were predominantly Grade 1–2. Grade 3–4 TRAEs were reported in 5 pts (2 each increased aspartate aminotransferase, increased weight; 1 each increased alanine aminotransferase, myalgia, hypersensitivity). There were no treatment discontinuations due to TRAEs. Conclusions: In this larger dataset with longer follow-up, larotrectinib continued to demonstrate rapid and durable responses, extended survival, and favorable long-term safety in pts with advanced TRK fusion lung cancer, including those with CNS metastases. These results support the wider adoption of next-generation sequencing testing for NTRK1–3 gene fusions in pts with lung cancer. Clinical trial information: NCT02576431 , NCT02122913 .

Larotrectinib (laro) long-term efficacy and safety in patients (pts) with tropomyosin receptor kinase (TRK) fusion thyroid carcinoma (TC).

6091 Background: Neurotrophic tyrosine receptor kinase ( NTRK) gene fusions are oncogenic drivers in a variety of tumor types, including TC. Laro is a first-in-class, highly selective, central nervous system (CNS)-active TRK inhibitor approved for tumor-agnostic use in pts with TRK fusion cancer based on a rapid, robust, and durable objective response rate (ORR) in both adult and pediatric pts with various cancers. Here, we report data on the subset of pts with TRK fusion TC treated with larotrectinib with long-term follow-up. Methods: Pts with TRK fusion TC enrolled in three laro clinical trials (NCT02576431, NCT02122913, NCT02637687) were included. Laro was administered at 100 mg twice daily to most pts; two pediatric pts received 100 mg/m2. Responses were assessed per independent review committee (IRC) using RECIST v1.1. Data cut-off: July 20, 2022. Results: As of data cut-off, 30 pts were eligible for efficacy assessment by IRC. The median age was 61.5 years (range 6–80) and the median time since initial cancer diagnosis was 5.0 years (range 0–46). The gene fusions involved NTRK1 (n =14; 47%) or NTRK3 (n =16; 53%). Fifteen pts (50%) received no prior systemic therapies, and six (20%) received ≥2; 23 (77%) received prior radioiodine. ORR was 63% (95% confidence interval [CI] 44–80): three (10%) complete response, 16 (53%) partial response (PR), five (17%) stable disease, four (13%) progressive disease, and two (7%) not evaluable. For pts classified as differentiated TC (DTC; n = 23), the ORR was 78% (95% CI 56–93). For pts classified as anaplastic TC (ATC; n = 7), the ORR was 14% (95% CI 0–58). All four pts with CNS metastases at baseline had a PR. Median time to response was 1.9 months, and median duration of response (DoR) was 43.3 months (95% CI 21.6–not estimable [NE]) at a median follow-up of 32.3 months. Median progression-free survival was 35.5 months (95% CI 23.4–NE) at a median follow-up of 34.0 months. Median overall survival (OS) was not reached (NR; 95% CI 48.7–NE) at a median follow-up of 46.4 months; the 48-month OS rate was 71% (95% CI 54–88). Median OS was NR (95% CI 48.7–NE) in DTC and 8.8 months (95% CI 2.6–NE) in ATC. At data cut-off, five pts who had disease progression continued treatment for ≥4 weeks due to continued clinical benefit. Treatment-related adverse events (TRAEs) were predominantly Grade 1–2. Grade ≥3 TRAEs were reported in two (7%) pts (anemia and decreased lymphocyte count). There were no treatment discontinuations due to TRAEs. Conclusions: Laro continues to demonstrate a rapid and durable response, extended survival, and a favorable safety profile in pts with TRK fusion DTC. Limited single-agent activity was observed in ATC. These results support the wider adoption of next generation sequencing panels, which include NTRK gene fusions, in pts with advanced TC prior to the initiation of any systemic therapy. Clinical trial information: NCT02576431 , NCT02122913 , NCT02637687 .

STUDY OF THE CONTRIBUTION OF GENES NERVOUS SYSTEM RELATED TO HEART FAILURE

Objective: Heart Failure (HF) is a complex syndrome that can be amplified by pathology associated with the nervous system. This study aims to investigate the contribution of genetic polymorphisms of Brain Derived Neurotrophic Factor (BDNF, rs6265), Neurotrophic Receptor Tyrosine Kinase 2 (NTRK2, rs2289656) and Neural Growth Factor (NGF, rs6330) genes in the development of HF. Design and method: A case-control study was conducted with a population of 261 individuals, of which 131 had HF (66 women and 65 men) with a median age of 77 years (Min = 31 years and Max = 96 years) and a BMI median of 25,00 Kg/m2 (Min = 17,80 Kg/m2 and Max = 47,30 Kg/m2), and 130 controls (113 women and 17 men) with a median of age 60,50 years (Min = 44 years and Max = 84 years) and a median BMI of 29,09 Kg/m2 (Min = 14,19 Kg/m2 and Max = 47,30 Kg/m2). Analyses of the 3 polymorphisms were performed using the Genotyping Endpoint PCR technique. Statistical analysis was performed using IBM software® SPSS® Statistics 28.0, with a statistical significance level set at p < 0,05. Results: For the rs2289656 polymorphism of the NTRK2 gene, the GG genotype was found to be a risk factor for HF [OR (CI, 95%) = 3.029 (1.053 – 8.718); p = 0.040]. The association between the presence of the A allele of the NTRK2 gene and HF [OR (CI, 95%) = 0.330 (0.115 – 0.950); p = 0.040] was also verified, with this allele constituting a protection factor. Regarding BDNF and NGF gene polymorphisms, no statistically significant differences were found. These results were adjusted for age, BMI and gender. Conclusions: These results show an association between the NTRK2 gene and HF, suggesting that this gene variation may enhance the development of HF. The identification of genetic polymorphisms that may somehow influence the development and severity of HF may allow its faster diagnosis and the application of methodologies for disease prevention. Moreover, our results will contribute to define a genomic profile associated with the role of the nervous system in HF.

Identification of Chimeric NTRK3 Genes in Papillary Thyroid Cancer Cells by Analyzing the Imbalance of the Expression of 5' and 3' mRNA Fragments.

The standard for detecting chimeric genes of neurotrophic receptor tyrosine kinases (NTRK) is next generation sequencing (NGS). However, this analysis is expensive and takes several days. As a rapid screening method for the detection of NTRK3-dependent papillary thyroid cancer, an analysis of the expression imbalance between 5' and 3' NTRK3 mRNA fragments was used (5'/3' RT-PCR). The reference method for detection of NTRK3 rearrangements was fluorescent in situ hybridization (FISH), and the most frequent rearrangements in papillary thyroid cancer were tested using reverse transcription PCR (RT-PCR). Using 5'/3' RT-PCR, 18 samples of papillary thyroid cancer carrying chimeric transcripts of NTRK3 mRNA were detected. The sensitivity of the developed technique was 88.9% and specificity was 99.3%. Thus, a fast and cost-effective method of screening samples of papillary thyroid cancer in paraffin blocks is proposed with acceptable sensitivity and specificity.

Gene co-expression network and differential expression analyses of subcutaneous white adipose tissue reveal novel insights into the pathological mechanisms underlying ketosis in dairy cows

Ketosis is a common nutritional metabolic disease during the perinatal period in dairy cows. Although various risk factors have been identified, the molecular mechanism underlying ketosis remains elusive. In this study, subcutaneous white adipose tissue (sWAT) was biopsied for transcriptome sequencing on 10 Holstein cows with type II ketosis [blood β-hydroxybutyric acid (BHB) >1.4 mmol/L; Ket group] and another 10 cows without type II ketosis (BHB ≤1.4 mmol/L; Nket group) at d 10 after calving. Serum concentrations of nonesterified fatty acids (NEFA) and BHB, as indicators of excessive fat mobilization and circulating ketone bodies, respectively, were significantly higher in the Ket group than in the Nket group. Aspartate transaminase (AST) and total bilirubin (TBIL), as indicators of liver damage, were higher in the Ket group than in the Nket group. Weighted gene co-expression network analysis (WGCNA) of the sWAT transcriptome revealed modules significantly correlated with serum BHB, NEFA, AST, TBIL, and total cholesterol. The genes in these modules were enriched in the regulation of the lipid biosynthesis process. Neurotrophic tyrosine kinase receptor type 2 (NTRK2) was identified as the key hub gene by intramodular connectivity, gene significance, and module membership. Quantitative reverse transcription PCR analyses for these samples, as well as a set of independent samples, validated the downregulation of NTRK2 expression in the sWAT of dairy cows with type II ketosis. NTRK2 encodes tyrosine protein kinase receptor B (TrkB), which is a high-affinity receptor for brain-derived neurotrophic factor, suggesting that abnormal lipid mobilization in cows with type II ketosis might be associated with impaired central nervous system regulation of adipose tissue metabolism, providing a novel insight into the pathogenesis underlying type II ketosis in dairy cows.

Green synthesized nitrogen-doped carbon quantum dots for the sensitive determination of larotrectinib in biological fluids and dosage forms: Evaluation of method greenness and selectivity

Recently, the kinase receptor inhibitor drug larotrectinib has been approved as a monotherapy for the treatment of patients with solid tumors containing the neurotrophic receptor tyrosine kinase gene fusion. In this paper, a novel sensitive spectrofluorimetric method was proposed for the determination of larotrectinib based on nitrogen-doped carbon quantum dots (N-CQDs) fluorescent probes. The proposed method is the first spectroscopic method for analysis of the cited drug, which is simple to implement and involves no pre-treatment steps or complicated techniques. The N-CQDs synthesis was performed by adopting a straightforward, fast, and environmentally friendly approach. It was achieved by means of a standard domestic microwave with inexpensive and readily available starting materials: orange juice (carbon source) and urea (nitrogen source). The synthesized N-CQDs were subjected to microscopic and spectroscopic characterization procedures. They were found to be stable with a sufficiently high fluorescence quantum yield (25.3%) and a small particle size distribution (2–5 nm). The motivation for the use of N-CQDs in this study arose from their excellent fluorescence intensities at 417 nm when excited at 325 nm. Larotrectinib was found to have a quantitative and selective quenching effect on the QDs fluorescence allowing for its sensitive determination. The drug’s quenching mechanism was investigated and found to be of the static type. Under optimal conditions, the proposed approach permitted the determination of larotrectinib over the concentration interval of 5.0–28.0 µg/mL. The method showed sufficient sensitivity with a detection limit of 0.19 µg/mL and a quantitation limit of 0.57 µg/mL, enabling the determination of LARO in spiked human plasma samples. The approach’s recovery percentage was found to be in the range of 99.09–100.73% for pure samples and 97.35–102.59% for plasma samples. The study also successfully applied the proposed approach to the commercial oral solution form of larotrectinib (Vitrakvi®) with high selectivity. Method greenness was further evaluated by adopting two metric tools, including the complementary green analytical procedure index (ComplexGAPI) and Analytical GREENNESS metric approach (AGREE), and it was confirmed to be excellent green. The proposed method was validated in accordance with the ICHQ2 (R1) recommendations and is considered an excellent candidate for potential application in the therapeutic monitoring of larotrectinib.

Japanese Society of Medical Oncology/Japan Society of Clinical Oncology/Japanese Society of Pediatric Hematology/Oncology-led clinical recommendations on the diagnosis and use of tropomyosin receptor kinase inhibitors in adult and pediatric patients with neurotrophic receptor tyrosine kinase fusion-positive advanced solid tumors

BackgroundClinical trials have reported the efficacy of tropomyosin receptor kinase (TRK) inhibitors against neurotrophic receptor tyrosine kinase (NTRK) fusion gene-positive advanced solid tumors. The accumulated evidence of tumor-agnostic agent has made since TRK inhibitors were approved and used in clinical practice. Therefore, we have revised the ‘Japan Society of Clinical Oncology (JSCO)/Japanese Society of Medical Oncology (JSMO)-led clinical recommendations on the diagnosis and use of tropomyosin receptor kinase inhibitors in adult and pediatric patients with neurotrophic receptor tyrosine kinase fusion-positive advanced solid tumors, cooperated by the Japanese Society of Pediatric Hematology/Oncology (JSPHO)’.MethodsClinical questions regarding medical care were formulated for patients with NTRK fusion-positive advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by JSCO, JSMO, and JSPHO voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and JSPHO, and the public comments among all societies' members was done.ResultsThe current guideline describes 3 clinical questions and 14 recommendations for whom, when, and how NTRK fusion should be tested, and what is recommended for patients with NTRK fusion-positive advanced solid tumors.ConclusionThe committee proposed 14 recommendations for performing NTRK testing properly to select patients who are likely to benefit from TRK inhibitors.